Published October 1, 1961
THE
IN
DISTRIBUTION
CONTRACTED
OF
MYOFIBRILS
FLUORESCEIN-LABELED
BERNARD
MUSCLE
TUNIK,
ANTIGENS
DETERMINED
BY
ANTIBODIES
Ph.D.,
and
HOWARD
HOLTZER,
Ph.D.
From the Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia.
Dr. Tunik's present address is Department of Biology, State University of New York, Long
Island Center, Oyster Bay
ABSTRACT
INTRODUCTION
Many properties of cross-striated myofibrils are
believed to be a function of the distribution of
the contractile proteins myosin, actin, and, to a
lesser extent, tropomyosin. For example, the
discontinuity of myosin molecules along the length
of the relaxed myofibrils is thought to determine
the cytology of the A band (1, 4, 5, 12, 15, 19).
Huxley and Hanson (4, 14, 15), on the basis of
salt extraction experiments and electron microscopy, have proposed that many characteristics
of the I band and the presence or absence of the
H band correlate, in rabbit muscle, with the distribution of actin along the myofibril. Similarly,
the sliding filament theory of contraction (13, 15)
proposes that overlap between thick (myosin?)
and thin (actin?) filaments observed in electron
micrographic sections is prerequisite to the interaction of the proteins responsible for shortening.
Clearly a better understanding of where in the
sarcomere the contractile proteins are localized
in different functional states should further our
understanding of how structure and function of
the myofibril are interdependent.
Fluorescein-labeled rabbit antibodies against
chicken myosin, light meromyosin (L meromyosin), heavy meromyosin (H meromyosin), actin,
and tropomyosin are each bound to discrete
regions of mature and embryonic sarcomeres
(12, 19). While there are unresolved problems
concerning the specificity and homogeneity of
these antisera--and of the antigens used in their
preparation--still their unique localization within
a sarcomere allows them to be used to trace these
antigens in myofibrils in different states. This
report describes the changes in the localization
of these antisera and, consequently, the changes
in the localization of the reacting muscle antigens
in relaxed, mildly contracted, and strongly contracted myofibrils.
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Chick myofibrils in different states of contraction were treated with fluorescein-labeled
antibodies. The rabbit antibodies were prepared against chick myosin, light and heavy
meromyosins, and actin. For any one state of contraction, a single myofibril was photographed through the phase contrast microscope, stained with one of the antiscra, and photographed through the fluorescence microscope. The cytological changes in the sarcomeres
accompanying contraction as observed under phase were correlated with changes in the
distribution of the precipitated antibodies as observed under the fluorescence microscope.
The changing patterns observed through the fluorescence microscope were compared with
those predicted by the sliding filament model of contraction.
Published October 1, 1961
MATERIA.L
AND
TABLE
METHODS
Glycerinated Muscle
Glycerol-extracted chicken breast muscle was
prepared as described by Szent-Gy6rgyi a n d Holtzer
(21). T o obtain relaxed muscle, bundles of muscle
fibers were tied to sticks at body length before excision. T o obtain contracted muscle, bundles were
allowed to contract freely after excision. R e l a x e d
a n d contracted m a t e r i a l was stored at - 15°C for at
least 2 weeks before use.
Stage
A
B
Myofibrils
A fiber b u n d l e was h o m o g e n i z e d in 10 ml M/60
PO4 buffer, pt-I 7.6, 25 per cent v/v glycerol (phosphate-glycerol), for 2 m i n u t e s at 0 ° C in an O m n i
m i x e r at 80 volts. T h e fibrils were t h e n w a s h e d three
times in 25 per cent phosphate-glycerol by centrifugation a n d resuspension.
Localization of Antibodies
68
D
Characteristic features
I b a n d : p r e s e n t . Z line : dense, faint, or u n d e t e c t a b l e . A b a n d : wide H b a n d .
I b a n d : p r e s e n t . Z line : d e n s e , faint, or u n d e t e c t a b l e . A b a n d : d i v i d e d by a d a r k c o n t r a c t i o n b a n d , t h e Cm b a n d .
T w o d a r k c o n t r a c t i o n b a n d s of u n e q u a l o p tical density. T h e l i g h t e r of t h e s e m a y be
f a i n t or u n d e t e c t a b l e . Fibrils b r e a k at t h e
lighter band.
Closely s p a c e d c o n t r a c t i o n b a n d s of e q u a l
o p t i c a l d e n s i t y ; o c c a s i o n a l l y lighter b a n d s
are also p r e s e n t .
images, often the widths of t h e two images did n o t
coincide. M o s t frequently a given fluorescent b a n d
a p p e a r e d slightly wider in p h o t o m i c r o g r a p h s taken
t h r o u g h t h e fluorescence microscope t h a n t h e s a m e
b a n d in p h o t o m i c r o g r a p h s t a k e n t h r o u g h the phase
microscope. At other times, however, the fluorescent
i m a g e was narrower. W h e t h e r this was d u e to the
distribution of labeled antibody, to the optics of the
fluorescence microscope, or to t h e p h o t o g r a p h i c
procedures is u n c e r t a i n (11).
RESULTS
A. Stages of Contraction
T h e d i f f e r e n t stages of c o n t r a c t i o n w e r e c h a r a c terized b y t h e p h a s e c o n t r a s t a p p e a r a n c e o f t h e
s a r c o m c r e s ( T a b l e I; Fig. l, line 1). A a n d I
b a n d s w e r e r e a d i l y r e c o g n i z e d in s t a g e A s a r c o m e r e s ; o c c a s i o n a l l y in r e l a x e d s a r c o m e r e s t h e
Z line w a s difficult to d e t e c t . S i m i l a r l y t h e A , I,
a n d Cm b a n d s o f s t a g e B w e r e r e a d i l y identified.
I n stages C a n d D , h o w e v e r , t h e A a n d I b a n d s
h a d d i s a p p e a r e d . As a c o n s e q u e n c e t h e Cm a n d
Cz b a n d s h a d to be i d e n t i f i e d b y o t h e r m e a n s .
The following evidence, adopting the terminology
of H o d g e (6), i n d i c a t e d t h a t in s t a g e C fibrils
t h e l i g h t c o n t r a c t i o n b a n d w a s t h e Ca b a n d a n d t h e
d a r k c o n t r a c t i o n b a n d w a s t h e Cm b a n d :
1. S t a g e A a n d B fibrils f r a g m e n t e d at o r n e a r
t h e Z line, n e v e r in t h e A b a n d . S t a g e C fibrils
f r a g m e n t e d a t or n e a r t h e l i g h t c o n t r a c t i o n b a n d .
2. I n stages A a n d B t h e a p p e a r a n c e o f t h e Z
line v a r i e d f r o m p r o n o u n c e d to u n d e t e c t a b l e .
T h i s also c h a r a c t e r i z e d t h e l i g h t c o n t r a c t i o n b a n d
in s t a g e C fibrils.
3. W h e n s t a g e A a n d B fibrils w e r e t r a n s f e r r e d
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY • VOLUME 11, 1961
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T h e fluorescein-labeled antibodies against chicken
myosin, L a n d H m e r o m y o s i n , a n d actin were those
used in previous studies (3, 10, 12, 19). A drop of
phosphate-glycerol suspension of myofibrils was
placed on a microscope slide. T h e fibrils were treated
with labeled antibody for 10 m i n u t e s by d r a w i n g a
drop of a solution of a n t i b o d y in phosphate-glycerol
u n d e r the coverslip. U n b o u n d antibody was r e m o v e d
by continuously d r a w i n g phosphate-glycerol u n d e r
the coverslip for 10 minutes. Both phase contrast a n d
fluorescence p h o t o m i c r o g r a p h s of the s a m e myofibril
were taken at a final magnification of 1000 times
before a n d after exposure to the different antibody
solutions. Control myofibrils were treated with
fluorescein-labeled n o r m a l g a m m a globulin by the
s a m e procedures. Fluorescence p h o t o m i c r o g r a p h s
were taken on spectroscopic plates ( K o d a k 103a G),
using a n O s r a m H B O 200 arc as a light source, 1 m m
C o r n i n g 5840 plus 1 m m Schott U G 1 as exciting
filters, a n d 1 m m each of W r a t t e n K 2 a n d 2B as
barrier filters. A n alternative a r r a n g e m e n t consisted of 6 m m Schott BG 12 exciting filters a n d 1 m m
each of Zeiss Sp o r a n g e 2 a n d Sp yellow. A Cardioid
condenser, N.A. 1.2, was used.
T h o s e regions of t h e sarcomere to w h i c h labeled
antibody was b o u n d could be identified by their
increased optical density u n d e r the phase contrast
microscope (7, 21), or by their emission u n d e r the
fluorescence microscope. T h e detection of fluorescence
was by far the m o r e sensitive m e t h o d . T h e phase contrast p a t t e r n of a n u n t r e a t e d sareomere was c o m p a r e d with the fluorescent p a t t e r n of the s a m e sarcomere after t r e a t m e n t with labeled antibody.
A l t h o u g h t h e centers of fluorescent images coincided
with the centers of corresponding phase contrast
I
Morphological Characterization of the Various
Stages of Contraction
Published October 1, 1961
FIGURE 1
to phosphate-glycerol from low salt solutions, the
greatest swelling occurred at the center of the
sarcomere. When stage C fibrils underwent the
same change in medium, the greatest swelling
occurred in the region of the dark contraction
band.
For stage D sarcomeres the dark contraction
bands were the Cz bands, the Cm bands being faint
or undetectable under phase. This identification
was based on the fact that in stage D sarcomeres
the fibrils fragmented at or near the dark contraction band and the greatest swelling occurred in
the region between the dark bands.
B. Distribution of Fluorescent Antibodies in
Different Stages of Contraction
I. Localization of Normal Globulin: There was
little, if any, binding of fluorescein-labeled normal
globulin by the myofibrils. W h e n present, the
fluorescence was variable and too weak to be
photographed.
II. Localization of Antimyosin: Stage A: As illustrated in Fig. 1, line 2, and Figs. 2 and 3, two
fluorescent patterns were encountered: a uniformly fluorescent A band, and a fluorescent A
band with an intensely fluorescent central stripe.
Note how the bound labeled antibody enhanced
the phase density of the H band in Fig. 3.
Stage B: The A band fluoresced throughout its
length, with the exception of a central region coincident with the Cm band. Note how the local precipitation of antibody has increased the phase density of the A band near the A-I junction (Fig. 4).
Stage C: The regions between the Cm and Cz
bands were fluorescent. The non-fluorescent band
bisecting the sarcomere was broader, and the
non-fluorescent Cz region narrower, than in stage
B. Consecutive A bands appeared to be converging on the non-fluorescent Cz band between them
to form the fluorescent Cz band described in the
next stage (Fig. 5).
Stage D: Only the regions coincident with the
dark contraction band, the Cz band, fluoresced.
Frequently the doublet nature of the fluorescent
Cz band could be observed along the edges of
the myofibril. The doublet nature of the fluorescent Cz band was often revealed by the terminal
bands where the fibril broke, which were one-half
the width of other C~ bands (Fig. 6).
III. Localization of Anti-L Meromyosin: Stage A:
The A band fluoresced throughout its length,
with the exception of a non-fluorescent band in
the center (Fig. 7).
B. TUNIK AND HOWARD HOLTZmR Muscle Antigens in Contracted Myofibrils
69
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Diagrammatic representation of the phase contrast appearance of myofibrils, in various stages of
contraction, and of the corresponding fluorescent patterns. No effort has been made to correlate
the changes of sarcomere pattern with changes of sarcomere dimensions. Diagonal lines indicate
reduced density under the phase contrast microscope and reduced fluorescence under the fluorescence
microscope. Arrows indicate the regions of the sarcomere where fibrils fragment. Line 1, phase contrast; line 2, antimyosin; line 3, anti-L meromyosin, line 4, anti-H meromyosin; line 5, actin.
Published October 1, 1961
!~ TO 6
Fibrils before and after treatment with antimyosin. In each figure there is a phase contrast photomicrograph of the untreated fibril (upper), a phase contrast photomicrograph of the same fibril
after treatment with labeled antibody (middle), and a fluorescence photomicrograph of the treated
fibril (bottom). Figs. 2 and 3, stage A. Fig. 4, stage B. Fig. 5, stage C. Fig. 6, stage D. See text for
details. X 2000.
Stage B: T h e non-fluorescent Cm b a n d was
b r o a d e r t h a n the corresponding Cm b a n d of stage
B sarcomeres treated with antimyosin. As a result,
a fluorescent doublet centered o n the Z region
was already indicated in stage B sarcomeres
treated with a n t i - L meromyosin (Fig. 8).
Stage C: O n l y the regions between the contraction bands fluoresced. T h e non-fluorescent C~
b a n d was quite narrow, the non-fluorescent Cm
b a n d quite broad. T h e doublet n a t u r e of the
fluorescent b a n d s converging o n the Z line was
most clearly seen in this stage (Fig. 9). Note the
70
deposition of a n t i b o d y observable u n d e r the phase
microscope a r o u n d the Cz region. T h o u g h considerable changes between stages B a n d C were
observed u n d e r phase, the fluorescent p a t t e r n s
were quite similar.
Stage D: T h e regions coincident with the Cz
b a n d s fluoresced. These fluorescent C~ b a n d s
exhibited the doublet n a t u r e described for antimyosin-treated fibrils (Fig. 10).
IV. Localization of Anti-H Meromyosin: Stage A:
O n l y the A b a n d fluoresced. T h e fluorescence
was most intense in the central portion of the A
THE JOURNAL OF BIOPHYSICAL AND BIOCHEMICAL CYTOLOGY VOLUME 11, 1961
•
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FIGURES
Published October 1, 1961
TO 10
Fibrils before and after treatment with anti-L meromyosin. Arrangement of phase and fluorescent
photomicrographs as before. Fig. 7, stage A. Fig. 8, stage B. Fig. 9, stage C. Fig. 10, stage D. See
text for details. X 2000.
band, in the region corresponding to the H band.
A broad, less intensely fluorescent area corresponded to the region of the A band near the A-I
junction. This latter region of reduced fluorescence
appeared to be the region stained by L meromyosin antisera (Fig. 11).
Stage B: The major difference between stage
A and stage B was a narrower non-fluorescent
I band. It is worth stressing that with the a n t i - H
meromyosin sera it was the Cm band which
fluoresced most strongly, whereas with antibody
to myosin or L meromyosin the Cm band was
non-fluorescent (Fig. 12).
Stage C: Again the Cm band fluoresced prominently and the Cz band was non-fluorescent. The
less fluorescent broad zones were often separated
from the fluorescent Cm band by narrow nonfluorescent regions. The broad zones of reduced
fluorescence corresponded to the regions rendered
fluorescent by the L meromyosin antisera (Fig. 13).
Stage D : The most fluorescent band lay midway
between the Cz bands. Adjacent portions of the
broad, less fluorescent bands formed a doublet
around the Cz area (Fig. 14).
V. Localization of Antiactin: Stage A: T h e I
band and particularly the H band fluoresced.
The fluorescence of the former was diffuse, that
of the latter sharp (Fig. 15).
Stage B: Similar to stage A, but the bands were
closer together. The bright, sharply defined
fluorescent bands corresponded to the Cm bands
observed under the phase microscope (Fig. 16).
Stage C: T h e regions corresponding to the
contraction bands as observed under the phase
microscope fluoresced. The sharp fluorescent band
corresponded to the Cm band, the more diffuse
zone to the Cz area (Fig. 17).
Stage D: The prominent fluorescent band lay
midway between the Ca bands. The dimmer
fluorescent band was coincident with the C~ bands.
DISCUSSION
Before interpreting the localization of the antisera
in contracted myofibrils it is worth while to review
B. TUNIK AND HOWARD [IoLTZER Muscle Antigens in Contracted Myofibrils
71
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FXOURES 7
Published October 1, 1961
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FIGURES 11 TO 14
Fibrils before and after treatment with anti-H meromyosin. Arrangement of phase and fluorescent
photomicrographs as before. Fig. 11, stage A. Fig. 12, stage B. Fig. 13, stage C. Fig. 14, stage D.
See text for details. X 2000.
their status as specific staining reagents. T h a t the
antimyosin molecules do combine selectively with
myosin as defined by other approaches is demonstrated by the following. Myosin antibodies (a)
complex exclusively with antigens in the A band;
(b) do not react with antigens in sperm tails,
cilia, mitotic figures, fibroblasts, or other types
of cells (3, 8, 9, 10, 23); (c) protect the A band
from salt solutions known to extract myosin (7,
21), and quantitatively prevent the extraction of
72
THE JOURNAL o F
BIOPHYSICAL
myosin from suspensions of mature and embryonic
fibrils pretreated with the antibody (8). What
is still unknown, however, is whether the myosin
antisera contain antibodies to A band antigens
other than myosin.
The localization of anti-L meromyosin on the
myofibril and its cross-reaction with myosin in
solution or in gel diffusion tests indicate that it
could be directed to a region of the myosin molecule (8, 19).
AND BIOCHEMICALCYTOLOGY
° VOLUME
11, 1961
Published October 1, 1961
TO
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FIGURES 15
17
Fibrils before and after trcatmcnt with antiactin. Arrangcment of phase and fluorcsccnt photomicrographs as before. Fig. 15, stage A. Fig. 16, stage B. Fig. 17, stage C. Scc text for details. X 2000.
The nature of the antigens binding a n t i - H
meromyosin is less clear. F r o m its localization in
relaxed and contracted myofibrils it is probable
that a n t i - H meromyosin solution contains at
least two components: one against antigens in the
regions of the A band near the A-I junction (possibly the L meromyosin antigens), and one against
antigens concentrated in the H band. The separation of the diffuse fluorescent band from the
fluorescent Cnl band in contracted fibers and the
similar staining of this region with anti-L meromyosin suggests that one component is shared by
the two meromyosin antisera. It is of interest that
analysis by means of Oudin tubes independently
suggests that one component is shared by the two
meromyosin antisera (19). Whether the H band
antigens correspond to a discrete fragment of the
myosin molecule exposed by trypsin digestion
(20), or whether it is against a still undefined
antigen, such as cholinesterase (2, 18, 22), is
not known at present.
Likewise there is evidence that the antiactin
sera contain at least two species of antibody
molecules: antibody precipitated by antigens in
the I band, and the component directed against
the H band antigens. The common behavior of
the H band and the Cm band when fibrils in
different stages of contraction are treated with
antiactin and a n t i - H meromyosin suggests that
the two solutions share a common population of
B. TUNIK AND HOWARD HOLTZER Muscle Antigens in Contracted Myofibrils
73
Published October 1, 1961
encroaches on the common Z line (see 16, 17).
Presumably the changes responsible for contraction result in the observed discontinuous distribution of myosin. This could be due either to a
" m o v e m e n t " of myosin away from the nonfluorescent regions or to an alteration in the availability of antigenic sites on the myosin molecule.
In either case these findings suggest a type of
heterogeneity in the A band that as yet cannot be
equated with the thick filaments observed under
the electron microscope.
The fluorescence patterns obtained with actin
and H meromyosin antisera also merit attention.
They suggest that (a) the I band antigens (revealed by antiactin sera) and the H band antigens
(revealed by antiactin and a n t i - H meromyosin
sera) maintain their relative positions throughout
all stages of contraction, and (b) a discontinuous,
non-overlapping distribution of these antigens,
or at least of their available antigenic sites, persists throughout all stages of contraction. These
findings would not have been anticipated by the
sliding filament model of contraction. Further
characterization of muscle antigens and their
antibodies should lead to a better correlation between the structure of myofibrils revealed by
electron microscopy and that revealed by the
fluorescence antibody technique.
This investigation was supported by Research Grants
B-493 and C-1957 from the National Cancer Institute
and the National Institute of Neurological Diseases
and Blindness of the National Institutes of Health,
United States Public Health Service, and by National Science Foundation grant G-14123. Dr.
Holtzer is a Scholar in Cancer Research of the
American Cancer Society.
Received for publication, March 23, 1961.
REFERENCES
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and HASS, G. M., J. Exp. Med., 1951, 94, 9.
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7. HOLTZER, H., Exp. Cell Research, 1959, Suppl. 7,
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THE JOURNAL OF BIOPHYSICALAND BIOCHEMICALCYTOLOGY - VOLIYME 11, 1961
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antibody molecules. This finding is in keeping
with the observation that antibody reacting with
H band material may be selectively removed from
a n t i - H meromyosin sera by absorbing against a
crude fraction of MgCl~-precipitated actin
(Hohzer, Szent-Gyorgyi, and Abbott, unpublished
observations). The remaining component of the
antiactin solutions the component reacting with
the I b a n d - - i s unique to the antiactin. Whether
this component is against the whole actin molecule or a fraction or bound impurity of the actin
molecule, or whether it is against still another
unknown antigen in the I band is being investigated.
The preparation of glycerinated bundles of
muscle fibers and their subsequent homogenization may cause contraction and stretching of the
fibrils. As a resuh, a given sarcomere length could
be arrived at by any combination of contraction
(isometric or isotonic) and stretching (9). The
possibility of these changes renders uncertain
any exact relationship between stages of contraction and relative or absolute sarcomere length.
However, sarcomere length does seem to decrease
regularly from stage A to stage D. It is likely that
these stages of contraction occur in their lettered
sequence. Assuming that these stages are stopmotion samples from a continuous morphological
change, then the redistribution or changing state
of the muscle antigens responsible for shortening
may be deduced from the fluorescence p~tterns
characteristic of each stage.
Two aspects of the fluorescence patterns obtained with myosin antisera are of particular
interest in view of the sliding filament model of
contraction. These are: (a) the absence of fluorescence in the center of the sarcomeres in all contracted fibrils, and (b) the formation of doublet
C, bands as the myosin from adjoining sarcomeres
Published October 1, 1961
11. HOLTZER, H., and HOLTZER, S., Cornpt.-rend.
tray. Lab. Carlsberg, sb. chim., 1960, 31, 373.
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13. t{UXLEY, A., Prog. Biophysics and Biophysic.
Chem., 1957, 7, 257.
14. HUXLEY, H. E., and HANSON, J., Biochim. et
Biophysica Acta, 1957, 23, 229.
15. HUXLEY,H. E., and HANSON,J., in The Structure
and Function of Muscle, (G. E. Bourne,
editor), New York, Academic Press, Inc.,
1960, 1, 183.
16. JORDON, H. E., Am. J. Anat., 1920, 27, 1.
17. JORDON, H. E., Physiol. Revs., 1933, 13, 301.
18. KOVER, A., KOVACS, T., and KONIG, T., Acta
Physiol. Acad. Sc. Hung., 1954, 5, 383.
19. MARSHALL, J. M., HOLTZER, H., FINCK, H.,
and PEPE, F., Exp. Cell Research, 1959, Suppl.
7, 219.
20. SZENT-GY6RGYI, A. G., in The Structure and
Function of Muscle, (G. E. Bourne, editor),
New York, Academic Press, Inc., 1960, 2, 1.
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Biochirn. et Biophysica Acta, 1960, 41, 14.
22. VAROA, E., SZmETI, J., and KISS, E., Acta
Physiol. Acad. Sc. Hung., 1957, 11, 253.
23. WENT, H., and MAZIA, D., Exp. Cell Research,
1959, Suppl. 7, 200.
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B. TIINIK AND HOWARD HOLTZER Muscle Antigens in Contracted Myofibrils
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